αvβ3 (CD51/CD61) is a member of the integrin family of cell surface adhesion receptors. Over 20 different αβ integrin heterodimers exist, each with different tissue and ligand specificities. Normal tissue distribution of αvβ3 is generally limited to high levels of expression on osteoclasts, with lower levels observed on platelets, megakaryocytes, kidney, vascular smooth muscle, placenta, dendritic cells (Weiss et al., 2001), and in varying amounts on normal endothelium (reviewed in Horton, 1997). In contrast to α5β1, which binds to only fibronectin, αvβ3 binds to a wide range of RGD-containing integrin ligands, including but not limited to fibronectin, vitronectin, osteopontin, von Willebrandt factor, and fibrinogen (Horton, 1997).
αvβ3 integrin is a multifunctional cell surface receptor that has pleiotropic roles in normal cell growth and survival. αvβ3 integrin can also contribute to oncogenesis. Consistent with this, upregulation of αvβ3 expression has been observed on the endothelial cells of angiogenic vessels, and binding of αvβ3 to the basement membrane is a critical step in the angiogenesis induced by basic fibroblast growth factor and tumor necrosis factor-α (Friedlander et al., 1995). Expression of αvβ3 has also been implicated in tumor invasion, and it has been shown that αvβ3 binds matrix metalloproteinase-2 (MM-2) and presents MMP-2 on the surface of invasive carcinomas and on invasive angiogenic endothelial cells (Brooks et al., 1996; Silletti et al., 2001).
αvβ3 also regulates cell growth and survival, since ligation of this receptor can, under some circumstances, induce apoptosis in tumor cells (Kozlova et al., 2001). Furthermore, disruption of cell adhesion with anti-αvβ3 antibodies, RGD peptides, and other integrin antagonists has been shown to slow tumor growth (Chatterjee et al., 2000; Chatterjee et al., 2001; and Brooks et al., 1996). Finally, the selective upregulation of αvβ3 expression on tumor blood vessels is also being explored as the basis for imaging of neoplastic lesion, and the αvβ3-specific antibody LM609 has been successfully used for this purpose in vivo (Sipkins et al., 1998).
Novel molecules capable of binding with high specificity to αvβ3 integrin have potential utility in several applications, and as a consequence, αvβ3 has been a frequent target for drug discovery and selection of new binding ligands. Phage display technology, in which combinatorial peptide libraries are expressed on the surface of bacteriophages, has been used to select for peptide ligands capable of binding to αvβ3—yielding a wide range of RGD-containing peptide sequences capable of interacting at moderate affinity with αvβ3 and other integrins (Koivunen et al., 1994; Healy et al., 1995). Phage-displayed random peptide libraries have also been constructed and screened using framework proteins such as the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). This resulted in the identification of phage clones which could be used to stain human umbilical vein endothelial cells in a flow cytometric assay. However, the ability of the purified recombinant CTLA-4 protein to stain cells, and its cross-reactivity with other integrins was not reported (Hufton et al., 2000).
Fibronectin is a natural ligand of integrins, and it contains repeats of three types of domains. The tenth fibronectin type III domain (FNfn10) includes the RGD sequence in the loop connecting the F and G β-strands (FG loop) (Main et al., 1992). FNfn10 was developed as a scaffold for phage display of peptides because of its small size (94 residues), monomeric assembly, and ability to retain its global fold while exposed loops were randomized (Koide et al., 1998). In addition, FNfn10 lacks cysteine residues and requires no post-translational modification, allowing for large-scale bacterial expression. It has been shown that residues in the FG loop including the RGD sequence are highly flexible (Main et al., 1992; Carr et al., 1997) and this flexibility of the FG loop has been implicated as the origin of the ability of FNfn10 to interact with multiple integrins (Main et al., 1992). While the stability of monobodies makes them well suited for intracellular studies, there has been no prior use of monobodies to probe for modified FG loop or other loop sequences with enhanced binding affinity for and selectivity between integrins, particularly αvβ3 integrin.
The present invention overcomes these and other deficiencies in the art.